The overall objective of this proposal is to define the cellular mechanisms contributing to carcinogenesis of cholangiocytes, the epithelial cells lining the biliary system. Chronic inflammation of the biliary tree predisposes to the development of cholangiocarcinoma, a malignant neoplasm originating from cholangiocytes. Because of this relationship, our long term goal is to understand the mechanisms by which inflammatory mediators contribute to the development and progression of cholangiocarcinoma. We have recently generated striking preliminary data demonstrating that biliary epithelia in a pre-malignant inflammatory disease (primary sclerosing cholangitis), and even cholangiocarcinomas paradoxically express the potent apoptosis inducing ligand TRAIL (tumor necrosis factor related apoptosis inducing ligand). However, because the inflammatory mediator IL-6 (lnterleukin-6) up-regulates the anti-apoptotic protein Mcl-1 (myeloid cell leukemia-1), they are resistant to TRAIL cytotoxicity. TRAIL was also found to promote cell migration/invasion in cholangiocarcinoma cell lines in an in vitro assay. Based on these extensive preliminary data, we propose the CENTRAL HYPOTHESIS that the inflammatory cytokine IL-6 up-regulates Mcl-1, which in turn converts TRAIL signaling from a pro-apoptotic ligand into a mediator facilitating cancer progression. Our proposal has three SPECIFIC AIMS. First, we will test the hypothesis that IL-6 up-regulates Mcl-1 by both transcriptional and post-translational mechanisms involving STATS and Akt signaling cascades, respectively. Second, we will test the hypothesis that Mcl-1 inhibits TRAIL-mediated apoptosis by binding specific pro-apoptotic BH3 only domain proteins of the Bcl-2 family, thereby blocking the lysosomal pathway of cell death. Finally, we will test the hypothesis that when apoptosis is blocked, TRAIL facilitates cancer progression by stimulating cell invasion and migration. To address these questions, we have established models of cholangiocarcinoma cell lines that differentially express Mcl-1, and have become adept at complex biochemical and molecular approaches to dissect the intracellular mechanisms regulating IL-6 signaling. We have also developed an understanding of Mcl-1 structure and function relationships, and have become familiar with TRAIL signaling. The proposal is conceptually and technically innovative as it tests new concepts in TRAIL biology employing state-of-the-art technologies. Not only will the information emanating from these studies potentially help identify therapeutic strategies for the treatment and/or chemoprevention of cholangiocarcinoma, but it may also help identify treatment avenues for other cancers arising within an inflammatory background. [unreadable] [unreadable] [unreadable]